Thyroid cancer is the most common endocrine malignancy, with an alarmingly steady increase in the last few years. Causal factors remain ill-defined. TSH is the major regulator of the thyroid gland involved in thyroid growth, differentiation, and thyroid hormone secretion. Evidence from animal models and new epidemiological studies suggest that TSH signaling pathways are critical for tumor progression. Thus, fully understanding the mechanisms involved downstream of the TSHR might provide potential new targets for therapy. TSH acts via the canonical TSHR-Gs-tmAC unit to synthesize cAMP, a second messenger mediating the action of numerous hormones. tmACs are members of a large family of transmembrane proteins with a G- protein-dependent adenylyl cyclase activity responsible for transducing membrane receptor signaling. Soluble adenylyl cyclase (sAC), an evolutionarily conserved member of the adenylyl cyclase family, is a relatively newly discovered activity not modulated by G-proteins, but instead activated by bicarbonate and calcium. The premise of this proposal is based on the identification of a novel sAC/Cyclase-Associated Protein 1 (CAP1)/Rap1b complex. We will address its involvement in TSH biology in four integrated specific aims. In Aim #1 we will utilize FRET-based cAMP sensors to assess whether pharmacological and genetic (shRNA/CRISPR-Cas9) methods that interfere with sAC activity impact TSH-mediated cAMP levels; preliminary data indicate sAC is involved in TSH-mediated cAMP dynamics regulating a PKA-PDE4 unit. In Aim#2 we will assess the involvement of the sAC-CAP1 complex in TSH-mediated G1/S progression. Pharmacological (sAC inhibitors) and genetic (sh-sAC/shCAP1) intervention inhibited TSH-mediated proliferation. To assess a specific role for CAP1-associated sAC, we will use shRNA/CRISPR-Cas9 approaches to downregulate CAP1 in cells and we will perform rescue assays upon infection with lentivirus coding for sh- or Cas9-resistant full length WT and mutant E171R-CAP1, deficient in sAC binding. In Aim #3 we will address the involvement of a nuclear sAC pool in TSH-mediated G1/S progression. We will use a newly developed optogenetic tool incorporating a blue-light activated cyclase (bPAC) fused to a blue- emitting nanoluciferase (nLuc). Targeting the bPAC-nLuc construct to distinct compartments will allow us to directly test whether cAMP generated in the nucleus is able to rescue proliferation upon sAC downregulation. We will complement these cell studies by thyroid-specific deletion of sAC in mice and rescue mediated by ultrasound-guided thyroid injection of compartment-specific sAC and bPAC-nLuc viral suspensions. In Aim #4 we will exploit mass spectrometry approaches to identify new CAP1-associated proteins utilizing newly developed reagents and optimized protocols for CAP1-pull down assays. Our long-term goal is to provide mechanistic details underlying the novel sAC-CAP1-Rap1 complex and its involvement in TSH biology.